Epidermal growth factor receptor (EGFR) gene alteration and protein overexpression in Malaysian triple-negative breast cancer (TNBC) cohort

Introduction

Human breast cancers represent a heterogeneous group of tumors and can be classified into different molecular subtypes based on histology, cellular origin, mutations, metastatic potential, disease progression, therapeutic response and clinical outcome.¹ While significant progress has been made for the treatment and patient outcome of breast cancers, the treatment of triple-negative breast cancer (TNBC) still remains a challenge due to its aggressive characteristics and limited treatment options. TNBC is a group of breast tumors characterized by the absence of expression of estrogen receptors (ER), progesterone receptors and human epidermal growth factor receptor 2 (HER2) and lacks the benefit of specific therapy that targets these proteins.² This subtype of invasive breast cancer carries a poor disease-free survival and associated with poor clinical outcomes accounting overall for about 17% of all breast cancers.^3,4 Among patients diagnosed with resectable (stages I–III) TNBC who completed tri-modality therapy (surgery ± radiotherapy + adjuvant or neoadjuvant chemotherapy), as many as 50% of patients experience disease recurrence and on an average 37% die in the first 5 years following surgery.^5,6 When comparing the TNBC of the Asian with the western population the onset age is much lower at 40–50 among Asians compared to western population (average age of occurrence 60–70). The overall survival remains poor due to the common occurrence of early relapse and predominant localization of distal metastases in visceral organs. Currently, no specific targeted therapy approach is available for TNBC outside clinical trials warranting novel targeting therapy discovery.^5,7

The commonly used systematic therapeutic options for TNBC are limited to conventional cytotoxic chemotherapy, whereas non-TNBC such as hormone receptor-positive, or HER2-positive breast cancer benefit from anti-hormonal or HER2-targeted therapy. Current treatment strategies for patients with TNBC include targeting angiogenesis through vascular endothelial growth factor and proliferation signaling focusing on agents for DNA repair, either directly through (ADP-ribose) polymerase inhibitors or indirectly through DNA-binding or DNA-damage potentiation.⁸ There are many ongoing TNBC trails for combination therapy that include anti-EGFR agents due to its central role in TNBC oncogenesis, metastasis and treatment outcome. Moreover, anti-EGFR agents (gefitinib) inhibit phosphorylation in an in vitro setting on TNBC cell lines (BT20, HCC1937 and MDA-MB-231) resulting in cell cycle arrest indicating the possibility of using recently developed anti-EGFR agents in TNBC treatment.⁹

The EGFR gene is located on chromosome 7p11.2, which belongs to the ErbB family of receptor tyrosine kinases that includes a ligand-binding extracellular domain, transmembrane domain, and a cytoplasmic catalytic kinase domain. The kinase was implicated in various cancers owing to their signaling function in Ras-MEK-ERK, PI3K-AKT-mTOR and Src-STAT3 pathways.^10,11 The EGFR pathway contains well-established oncogenes including EGFR, KRAS, BRAF and PIK3CA genes that modulate gene activations in solid tumors including lung, colorectal cancer (CRC) and pancreatic ductal adenocarcinoma.^12–15 The EGFR and EGFR-like peptides are often over-expressed in human carcinomas, and in vivo and in vitro studies have shown these proteins induce cell transformation.¹⁶ Moreover, anti-EGFR therapies correlated with longer survival of patients with colon carcinoma, non-small cell lung cancer (NSCLC) and squamous-cell of head and neck carcinoma.¹⁷

Several clinical studies analyzing EGFR protein expression in various populations showed a wide variety of results ranging from 13% positive EGFR expression detected through immunohistochemistry (IHC) in Korea and Italy to a very high positive expression of 76% in Switzerland and 72% in France.^18–21 The EGFR autocrine pathway contributes to a number of processes pivotal in breast cancer development and progression, including cell proliferation and migration.²² Again gene amplification analyses from various countries showed a very high variation between 2% in German population to about 24% in Switzerland. Atypical EGFR activation in tumor cells can result from increased transcriptional expression, mutation and/or gene amplification.²⁰ The increased EGFR protein and transcript levels correlate with poor prognosis in various epithelial cancers, such as CRC, NSCLC and endometrial cancer.^23–25 In TNBC, EGFR expression is also associated with poor clinical outcome and aggressive metastasis in western population.¹⁹ It is therefore essential to profile for EGFR expression and variations in specific population to ascertain the possibilities of using EGFR as an independent prognosticator and anti-EGFR therapies.

Although the EFGR expression and gene alterations are extensively studied in the western population, the EGFR status in Malaysian TNBC cohort is completely lacking. Here, we report the EGFR copy number alteration and protein overexpression studied using fluorescence in situ hybridization (FISH) and IHC, respectively, in the representative Malaysian TNBC cohort to review the possibility of stratifying patients using EGFR status and using anti-EGFR therapy (Table 1).

Table 1 Summary of patient samples analyzed in the study

Materials and methods

Patients and tissue samples

Fifty-eight formalin-fixed paraffin-embedded (FFPE) satisfactory tissues of patients with TNBC from 2005 to 2013 were obtained from Universiti Kebangsaan Malaysia Medical Centre (UKMMC). The cases included in this study are the ones that are classified as invasive ductal TNBC based on the expression of standard biomarkers (ER, progesterone and HER-2), immunohistochemical data and those cases with complete clinical data.

Immunohistochemistry (IHC) analysis

Tissue sections with 4 µm thickness were cut from the FFPE blocks, dried, deparaffinized and rehydrated using standard operating procedures. The EGFR expression was detected using monoclonal mouse anti-human EGFR, wild-type (DAK-H1-WT) (Dako, Santa Clara, CA, USA) antibody and EnVision™ FLEX+ High pH kit (Dako). EGFR antibody was applied to a representative section of the TNBC cases as manufacturer’s protocols, with Proteinase K proteolytic epitope retrieval for 5 mins, followed by incubation with the primary EGFR antibody at room temperature for 30 mins. DakoCytomation Mouse IgG1 (Dako) was used as negative control and colon cancer was used as positive control. Both positive and negative controls were run simultaneously with the test samples. The slides were stained lightly with hematoxylin prior to scanning and viewing.

EGFR expression was scored by pathologist as following: 0, no staining or weak membranous staining in <10% of the tumor cells; 1+, weak membranous staining in ≥10% of the tumor cells; 2+, moderate membranous staining in ≥10% of the tumor cells; 3+, strong membranous staining in ≥10% of the tumor cells. Complete and incomplete membranous staining were both accepted and a score of 2+ onwards was considered to be EGFR overexpression (Table 2).

Table 2 Summary of FISH and IHC scores for the patient samples analyzed

FISH assay for the detection of EGFR gene alterations

FISH detection of EGFR gene alteration was performed by hybridizing slides with fluorescent-labeled dual-colored probes, XL EGFR amp (MetaSystem, Altlussheim, Germany) according to the manufacturer’s instructions. The probe mixture consists of an orange labeled probe which hybridizes to the EGFR locus at 7p11 and a green labeled probe which hybridizes to the 7cen region. The fluorescence hybridization signal was observed using fluorescence microscope and analyzed using CytoVision (Leica Biosystems, Nussloch, Germany) software.

For each sample, 50 non-overlapping tumor cells were evaluated. EGFR gene copy number was classified into normal disomy (≤2 copies in >90% of cells); low trisomy (≤2 copies in ≥ of 40% of cells, three copies in 10–40% of cells and ≥4 copies in <10% of cells); high trisomy (≤2 copies in ≥40% of cells, 3 copies in ≥40% of cells and ≥4 copies in <10% of cells); low polysomy (≥4 copies in 10–40% of cells); high polysomy (≥4 copies in ≥40% of cells) and EGFR gene amplification (presence of high EGFR gene clusters and a ratio of the EGFR gene to chromosome 7 of ≥2 or >15 copies of EGFR per cell in ≥10% of cells).²⁶ The cases were then classified into two major groups which were low EGFR copy number (disomy, low trisomy, high trisomy and low polysomy) and high EGFR copy number (high polysomy and gene amplification).

Statistical analysis

Statistical analysis was carried out using Statistical Package for Social Sciences (SPSS) software, version 10 (SPSS, Chicago, IL, USA). The Chi-square test was used to determine the relationship between EGFR gene alterations and EGFR protein expression. The result was considered to be statistically significant at a P<0.05.

Results

EGFR copy number alteration

In this study, FISH results were divided into low gene copy number and high gene copy number. Of 58 TNBC cases, gene amplification was shown in three (5.2%) cases while high polysomy in six (10.3%) cases. The 49 remaining cases showing disomy were detected in six cases (10.3%), low trisomy in 24 cases (41.4%), high trisomy in 14 cases (24.1%) and low polysomy in five cases (8.6%). The percentage of cells with EGFR gene amplification was evaluated in 50 non-overlapping cells showing abnormal signal patterns in ≥10% cells (Tables 2 and 3). Representative cases of low copy number and high copy number of EGFR gene are shown in Figure 1.